PCR for detection of Leishmania donovani from microscopically negative tissue smears of suspected patients in Gondar, Ethiopia

BackgroundAs untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are pivotal for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. MethodologyWe conducted a retrospective study on stored microscopically negative spleen and bone marrow smears from suspected VL patients collected at the Leishmaniasis Research and Treatment Center (LRTC) in Gondar, northern Ethiopia between June 2019 and November 2020. Sociodemographic, clinical and treatment data were collected and samples were tested by real-time PCR targeting kinetoplast DNA. Principle findingsAmong the 191 eligible samples (135 spleen and 56 bone marrow) with a microscopically negative and valid PCR result, 119 (62.3%) were positive by PCR, although Ct values for some were high (median 33.0). Approximately three quarters of these undiagnosed primary VL (77.3%) and relapse (69.6%) patients did not receive antileishmanial treatment. Of the 56 microscopically negative bone marrow samples, 46 (82.1%) were PCR positive, which is considerably higher compared to the microscopically negative spleen samples, for which 73 out of 135 (54.1%) were PCR positive. The odds of being PCR positive were significantly higher for bone marrow aspirates and higher when white blood cell values were lower and splenomegaly (in cm) was more pronounced. ConclusionsThis study demonstrates that a lot of suspected VL patients remain undiagnosed and untreated. This indicates the urgent need for better diagnostics for VL in the East-African region. The outcomes of PCR positive should be closely monitored and treatment should be provided if the patient deteriorates. In resource limited settings, implementation of PCR on bone marrow aspirate smears of patients with low WBC values and splenomegaly could lead to considerable improvements in patient management. Author summaryAs untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are important for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. To prove this, we conducted a study on stored microscopically negative spleen and bone marrow aspirate smears from suspected VL patients in Gondar, Ethiopia. Clinical and treatment data were collected and samples were tested for Leishmania by PCR. We found that about 60% of these microscopically negative samples were PCR positive. This PCR positivity rate was considerably higher in patients with a microscopically negative bone marrow compared to splenic aspirate. Importantly, more than three quarters of the patients with a PCR positive sample was not treated for VL. Overall, our study demonstrates the gap in the diagnostic algorithm for VL in northern Ethiopia, especially when bone marrow samples are used. In resource limited settings, we advise to challenge the current diagnostic algorithm and implement molecular tools to accurately diagnose patients. This could lead to considerable improvements in patient management in Ethiopia and beyond.